Blood is a highly dynamic system, in which billions of mature cells are replenished every day. It is traditionally thought that this ongoing process of cel regeneration exclusively depends on hematopoietic stem cells (HSC); whereas progenitors, the abundant progeny of HSCs, are generally thought to represent transient amplifying stages, where rare primitive cells are dramatically expanded before the final production of functional blood cells. While the essential role of HSC in blood regeneration has been repeatedly demonstrated in both experimental and therapeutic transplantation, recent studies using novel in situ lineage tracing techniques have revealed very limited contribution of HSC to blood cell generation at steady state under native, unperturbed conditions. In contrast, the progenitor populations, although non-transplantable, have a much more persistent ability for blood generation during native hematopoiesis. These findings collectively demonstrate a previously unappreciated level of regulation of blood homeostasis at the stage of progenitor cells. They also highlight the potential use of progenitors for robust hematolymphoid regeneration during bone marrow transplantation. This proposal aims to further characterize the cellular and molecular mechanisms by which the progenitors sustain their long-term in vivo hematopoietic activities. Aim 1 will attempt to elucidate the contribution by the different subsets of the progenitors in ongoing blood cell production. This will be achieved by comparing clonal overlapping between different blood lineages, and also by directly measuring the clonal output of the progenitor subsets at multiple time points following induction of transposon labeling. In Aim 2, the clonal complexity and clone size of progenitors will be examined at different stages of the mouse lifespan. Results of this analysis will help to determine if progenitor self-renewal is achieved at the level of single cells, or is accomplished through population dynamics. The role of cellular dormancy in preserving progenitor activity, and molecular basis of distinct clonal behaviors will be explored as well in this aspect of the study. Aim 3 will focus on the impact of bone marrow transplantation on functional properties of progenitors. The H2B-GFP label-retaining model will be used to test the global impact of transplantation on progenitor proliferation and survival. And the transposon technology will help compare the clonal behaviors of progenitors in self-renewal and differentiation during native and transplant hematopoiesis. In the aggregate, these proposed studies will bring new insights to the old questions of cellular mechanisms of blood regeneration. Outcomes of the proposed work may also provide clues for the design of better strategies for preserving progenitor activity during transplantation.